Regulation of the earliest stem and progenitor cells is not completely understood. In this context we propose to test the hypothesis that the combination of two early acting cytokines: Steel factor (SLF) and the Flt- 3/Flk-2-ligand, which respectively utilize the tyrosine kinase receptors c-kit and Flt-3-/Flk-2 for their action is important for proliferation and self-renewal of early stem/progenitor cells. This will be done by using recombinant(r) adeno-associated virus (AAV) vectors to obtain high efficiency stable integration of the genes for the soluble and membrane- bound forms of both ligands into high purified stem/progenitor cells. The aims are to: 1) construct high titer rAAV vectors containing soluble or membrane-bound forms of the human (hu) or murine (mu) ligands with different promoters and selectable markers; 2) evaluate different phenotypic target cell populations that are highly enriched for stem and/or progenitor cells (including hu CD34+++ cord blood and marrow cells, and their subsets distinguished by CD38 and/or HLA-DR antigens and mu Sca- 1+ marrow cells) for their responsiveness to transduction by separate AAV vectors containing SLF or Flt-3/Flk-2, alone and in combination, and also to evaluate procedures necessary for optimal transduction of these cells; and 3) determine the success of the different transduction procedures and viral vectors on the different phenotypic target cell populations by utilizing a variety of in vitro and in vivo assays to assess effects on proliferation, self-renewal and differentiation of the cells. Assays in vitro include those for high proliferative potential colony forming cells (HPP-CFC), and immature and mature subsets of multipotential (CFU-GEMM), erythroid (BFU-E), granulocyte-macrophage (CFU-GM), granulocyte (CFU-G), and macrophage (CFU-M) progenitors and for long-term culture-initiating cells (LTC-IC). Assays in vivo will utilize hu-cell inoculated sublethally irradiated SCID mice and mu-cell inoculated lethally irradiated syngeneic normal mice. Self-renewal in vitro and in vivo will respectively be estimated by replating capacity of individual colonies and transfer of marrow cells from l degree to 2 degrees mouse recipients. It is anticipated that these studies will help to clarify a role for SLF and Flt-3/Flk-2- ligand in early hematopoiesis and the use of rAAV vectors for gene transduction and future gene therapy.